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Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during antimicrobial photodynamic therapy
Accepted Manuscript Title: Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during antimicrobial photodynamic therapy Authors: Maryam Pourhajibagher, Hossein Kazemian, Nasim Chiniforush, Nava Hosseini, Babak Pourakbari, Atefeh Azizollahi, Faranak Rezaei, Abbas Bahador PII: DOI: Reference:
S1572-1000(18)30242-4 https://doi.org/10.1016/j.pdpdt.2018.09.014 PDPDT 1253
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
24-7-2018 17-9-2018 24-9-2018
Please cite this article as: Pourhajibagher M, Kazemian H, Chiniforush N, Hosseini N, Pourakbari B, Azizollahi A, Rezaei F, Bahador A, Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during antimicrobial photodynamic therapy, Photodiagnosis and Photodynamic Therapy (2018), https://doi.org/10.1016/j.pdpdt.2018.09.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during
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antimicrobial photodynamic therapy
Maryam Pourhajibagher1, Hossein Kazemian2,3, Nasim Chiniforush4, Nava Hosseini5, Babak Pourakbari6, Atefeh Azizollahi7, Faranak Rezaei8*, Abbas Bahador9
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Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 2 Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran. 3 Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 4 Laser Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 5 Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran. 6 Pediatric Infectious Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran. 7 School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. 8 Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. 9 Oral Microbiology Laboratory, Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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*Correspondence Address: Franak Rezaei, Ph.D., Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, PO Box:381351698, Khorramabad, Lorestan. Fax: + 986633120150. Email: [email protected] Highlights Curcumin (CUR) and indocyanine green (ICG) mediated antimicrobial photodynamic therapy (aPDT) have considerable antibacterial effects against Enterococcus faecalis. aPDT by CUR and ICG has more anti-biofilm formation activity against E. faecalis than toluidine blue O (TBO) and methylene blue (MB). The antimicrobial activities of CUR mediated aPDT are higher than TBO and MB.
Abstract
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Background: Despite the high success rate of endodontic treatment, failure may occur in some cases. In this case, Enterococcus faecalis is the most common species in endodontic treatment failure and post-treatment apical periodontitis. Therefore, a new adjunctive strategy is needed for the prevention of endodontic infections due to E. faecalis. The aim of the present study was to compare the antimicrobial and anti-biofilm activities of different common photosensitizers (PSs) for use in antimicrobial photodynamic therapy (aPDT) against E. faecalis. Materials and methods: E. faecalis strain ATCC 29212 was used as the tested strain and methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG), and curcumin (CUR) were used as PSs. Irradiation was carried out using diode laser and light emitting diode (LED) at wavelengths related to the above PSs. Then, antimicrobial and anti-biofilm activities were measured using the microbial viability assay and crystal violet test, respectively. Results: aPDT with using the above PSs significantly decreased the CFU/mL count of E. faecalis compared to the control group (P < 0.05). The killing percentage of E. faecalis via PS mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0% for CUR, ICG, TBO, and MB, respectively. aPDT using the above PSs significantly decreased the biofilm formation ability of E. faecalis compared to the control group (P < 0.05). The biofilm reduction percentage of the PSs was 68.4%, 62.9%, 59.0%, and 47.6% for CUR, ICG, TBO, and MB, respectively. Conclusion: CUR and ICG mediated aPDT exhibited considerably more antimicrobial activity than other PSs, while TBO and MB demonstrated weaker anti-biofilm effects against E. faecalis compared to other PSs. Keywords: Enterococcus faecalis; antimicrobial photodynamic therapy; methylene blue; toluidine blue O; indocyanine green; curcumin
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1. Introduction Despite the high success rate of endodontic treatment, failure may occur in some cases. One of the important causes of endodontic failure is secondary/persistent endodontic infection in the root canal system, maybe due to evasion of disinfectants at the time of root canal treatment obturation [1]. In this regard, Enterococcus faecalis is the causative agent in 20-70% of endodontic failures due to its ability to form biofilms as well as its high resistance to endodontic medicaments [2-5]. Following the emergence of resistant E. faecalis to antimicrobial agents isolated from root canals, prevention of secondary endodontic infection caused by this bacterium has become very complicated [6]. Efforts in this regard have led to the investigation, development, and setting up of novel antimicrobial strategies. Antimicrobial photodynamic therapy (aPDT) is a novel antimicrobial approach. aPDT acts through generation of reactive oxygen species (ROS) in the presence of molecular oxygen by using a combination of a photosensitizer (PS) and light at a specific wavelength. A PS is a nontoxic dye that binds to the outer surface, cell membrane, and DNA of the microorganism, and becomes activated following irradiation with light. aPDT causes bacterial cell death by microbial cell envelope disruption and DNA damage [7]. Many studies have
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demonstrated the antimicrobial properties of aPDT against local infections [8, 9]. Moreover, it has been shown that aPDT not only can be used for killing bacteria, but it can also be applied to reduce the impact of bacterial virulence factors such as biofilm formation and prevent bacterial colonization in the root canal [10]. Different types of PSs have been used for endodontic treatment in recent years [11]. Toluidine blue O (TBO) and methylene blue (MB) are amphiphilic, positively charged PSs that have a low molecular weight. They penetrate into the outer membrane through binding to microbial surface proteins. These PSs have bactericidal effects against both Gram-positive and Gram-negative bacteria. The absorption of TBO and MB peaks at 635 and 660 nm, respectively [11]. Indocyanine green (ICG) is an anionic PS with antimicrobial properties mainly in photothermal versus photochemical therapy and an absorption peak at 810 nm [11]. Curcumin (CUR), another PS, is a phenolic compound capable of absorbing light at 450 nm [12]. To the best of our knowledge, no study has compared the effect of aPDT based on four common types of PSs against bacterial pathogens. Considering the role of E. faecalis as a leading cause of persistent/secondary endodontic infection, the aim of the present study was to compare the potency of antimicrobial and anti-biofilm effects of TBO-, MB-, ICG-, and CUR-aPDT against E. faecalis to find a better endodontic disinfection procedure.
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2. Material and Methods 2.1. Bacterial strain E. faecalis strain ATCC 29212 was obtained from the Iranian Biological Resource Center (Tehran, Iran) and aerobically grown in the fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37 °C until logarithmic growth phase at a final concentration of 1.0 × 106 colony forming units (CFU)/mL, verified by spectrophotometry at 600 nm.
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2.2. Photosensitizers and light sources Based on the results of previous studies, the maximum concentration of PSs that not causing cytotoxicity and tooth coloration was used [8, 13]. Stock solutions of TBO and MB (SigmaAldrich, Steinheim, Germany) were prepared in sterile 0.9% (wt/vol) NaCl at a final concentration of 0.1 mg/mL. A stock solution of ICG was prepared by dissolving ICG powder (Santa Cruz, USA) in 1.0 mL sterile distilled water at a final concentration of 1.0 mg/mL. A stock solution of CUR (Sigma-Aldrich, Steinheim, Germany), at a final concentration of 5.0 mg/mL, was prepared using a non-toxic concentration of dimethyl sulfoxide (0.5%) [13]. Then, all of the stock solutions were sterilized using a 0.22-nm millipore filter and kept in dark conditions before use. A diode laser at wavelengths of 635 nm (Klas DX62, Konftec, Taiwan), 660 nm (Klas DX62, Konftec, Taiwan), and 810 nm (Klas DX82, Konftec, Taiwan) was used for TBO, MB, and ICG, respectively. A light emitting diode (LED) (DY400-4, Denjoy, China) at a wavelength of 450 nm was applied for activation of CUR (Table 1). According to previous studies, the maximum light exposure time not toxic for eukaryotic cells was used in this study (i.e. 2, 5, 5, and 5 minutes with an energy density of 32.5, 117.18, 171.87, and
300-420 J/cm2 at a wavelength 810, 660, 635, and 450 nm for ICG, MB, TBO, and CUR, respectively; Table 1) [8, 13]. The flowchart of the experiment steps is shown in Figure 1.
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2.3. Study design To evaluate the antimicrobial and anti-biofilm activities of different PSs for use in aPDT against E. faecalis, the experimental groups containing E. faecalis strains (n=39) were subjected to the following: A: PS alone: A1: TBO (n=3) A2: MB (n=3) A3: ICG (n=3) A4: CUR (n=3) B: Radiation alone B1: Diode laser at 635 nm (n=3) B2: Diode laser at 660 nm (n=3) B3: Diode laser 810nm (n=3) B4: LED (n=3) C: aPDT (PSs plus corresponding light) C1: TBO plus diode laser at 635 (n=3) C2: MB plus diode laser at 660 (n=3) C3: ICG plus diode laser 810 (n=3) C4: CUR plus LED (n=3) D: Control group: Bacterial suspension with no treatment (n=3)
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2.4. Evaluation of aPDT against E. faecalis in planktonic form In PS alone groups, 100 µL of the 1.5×106 CFU/mL bacterial suspension was added to each well of a 96–well flat-bottomed microtiter plate (TPP, Trasadingen, Switzerland). Then, 100 µL of each PS was added to the bacterial suspension separately. For bacterial cells to absorb the photosensitizer, the mixture was incubated at room temperature (25 ± 2 oC) in the dark for 5 min. In the radiation alone group, 200 µL of the 1.5×106 CFU/mL bacterial suspension was added to each well of the microtiter plate. The bacterial suspension was then exposed to the light source according to the wavelengths mentioned earlier. aPDT was carried out according to a previous study [8]. Briefly, 100 μL of the 2X BHI broth was added into each well of 96-well flat-bottomed sterile polystyrene microplates. Then, 100 μL of each PS solution was added to each well using a multi-channel pipet. In the next step, 100 μL of the 1.5×106 CFU/mL bacterial suspension was added to each well. The microplates were incubated in a dark room for 5 min. Then, the bacterial suspensions were exposed to radiation corresponding to each PS. In this regard, radiation was done at room temperature (25 ± 2 °C) for 2, 5, 5, and 5 min at 810, 660, 635, and 450 nm for ICG, MB, TBO, and CUR, respectively. A bacterial suspension without any treatment was used as the control group.
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The laser and LED probes were fixed 1 mm above the top surface of the microplate by a microphone stand. To prevent beam reflection from the tabletop during aPDT, a black paper was used under the microplates. Then, after aPDT, for determination of CFU/mL of the bacterial cells, 10 μL aliquots of each well was cultured on BHI agar plates (Merck, Darmstadt, Germany) and incubated at 37 °C for 24 h. The number of CFUs/mL was determined after incubation according to a method proposed by Miles and Misra [14].
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2.5. Evaluation of aPDT against biofilm formation ability of E. faecalis To evaluate the anti-biofilm activities of aPDT mediated by the aforementioned PSs, biofilms of E. faecalis were formed using 12-well flat-bottomed sterile polystyrene microplates according to previous studies with minor modifications [15, 16]. Briefly, 2000 μL of the 1.5 × 108 CFU/mL bacterial suspension was treated as mentioned earlier. Aliquots of the bacterial suspension were grown on polyester coverslips (Thermo Scientific™ Nunc™ Thermanox™, GmbH, Germany) at 37 °C for 24 h. The coverslips were transferred to the new sterile microplates and gently washed using 2000 μL phosphate-buffered saline (PBS, pH: 7.4). In the new sterile microplates, each coverslip was then stained with 0.1% (w/v) crystal violet for 20 min at room temperature, and then washed twice with distilled water. The biofilm was solubilized using 1000 µL of 95% ethanol for 20 min. Then, 100 µL of the content of each well was transferred onto 96-well flat-bottomed sterile polystyrene microplates, and the optical density of each well was quantified at 570 nm using a microplate reader (Thermo Fisher Scientific, US). A bacterial suspension without any treatment was used as the control group.
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2.6. Statistical analysis The results were analyzed with one-way analysis of variance (ANOVA) and Tukeys’ test using the SPSS software version 22.0. All experiments were performed in triplicate. The results are reported as mean ± standard deviation (SD). P values less than 0.05 were considered significant.
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3. Results As shown in Figure 2, aPDT mediated by the aforementioned PSs significantly reduced CFU/mL count of E. faecalis compared to the control group (P < 0.05). CUR, ICG, TBO, and MB mediated aPDT decreased the CFU/mL by 250, 56, 7, and 3 folds compared to the control group, respectively. The killing percentage of E. faecalis by CUR, ICG, TBO, and MB mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0%, respectively. According to our data, CUR-aPDT reduced the CFU/mL count of the E. faecalis almost equal to ICG-aPDT but more than TBO- and MBaPDT (P < 0.05, Figs. 2 and 3). According our data in Table 2, treatment with each PS (MB, TBO, ICG, and CUR), and radiation alone (diode laser at 635, 660, and 810 nm as well as LED) caused no significant CFU/mL reduction in E. faecalis. In this regard, CUR and ICG mediated aPDT had more antimicrobial activity than TBO- and MB- aPDT. The biofilm reduction percentage of CUR, ICG, TBO and MB mediated aPDT was 68.4%, 62.9%, 59.0%, and 47.6%, respectively (P < 0.05; Fig. 4). Based on the results of SEM images, aPDT mediated by the above PSs disrupted the biofilm structure of E. faecalis more than other groups
(Fig. 5). In addition, as shown in Figure 4, 5 and Table 2, treatment with a PS alone, diode laser alone at different wavelengths, and LED alone caused no significant reduction in the formation of E. faecalis biofilms.
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4. Discussions aPDT is considered a minimally invasive method for treatment of local infections, such as endodontic infection [17, 18]. In the present study, the efficacy of aPDT mediated by different common PSs against planktonic and biofilm growth of E. faecalis was tested. E. faecalis was selected as the test strain because it has been found to colonize in the root canal and cause different disorders [19, 20]. Pourhajibagher et al. showed that ICG decreased the cell count and biofilm formation of Porphyromonas gingivalis to 30.4% and 25.1%, respectively [21]. In the present study, TBO showed high yet non-significant antimicrobial and anti-biofilm activities, more than MB, in PS mediated aPDT against E. faecalis. Our data showed that the killing percentage of E. faecalis by TBO mediated aPDT was 85.1%. The results of this study showed that neither LED nor diode laser nor PSs, when used alone, had antimicrobial and anti-biofilm properties, which was consistent with the results of studies conducted by Pourhajibagher et al. [8], López-Jiménez et al. [22], and Rödig et al. [23]. CUR had more (but non-significant) antimicrobial and anti-biofilm formation activities compared to other PSs. It has been reported that there is an assembly of a protein-filamenting temperaturesensitive mutant Z (FtsZ). The proto-filaments are inhibited by CUR, which leads to an increase in the GTPase activity of FtsZ. Increased GTPase activity due to the FtsZ assembly causes bacterial cell death [24]. Our data showed that CUR-aPDT (5.0 mg/mL, 5 min radiation) had the highest bactericidal and anti-biofilm activities against E. faecalis. The antimicrobial activity of CUR mediated aPDT has been evaluated in different bacterial species. Paschoal et al. showed that simultaneous use of blue light LED plus CUR had a better antimicrobial effect on Streptococcus mutans, which was consistent with our results [25]. Najafi et al. reported that CUR is an effective substance in preventing the growth of Aggregatibacter actinomycetemcomitans, the impact of which is reinforced when used simultaneously with an aPDT method [13]. However, some studies have reported different results compared to our analysis. Lozano et al. evaluated the photodynamic effect of MB, Rose Bengal (RB), and CUR in combination with white light against S. mutans, S. sanguis, and Candida albicans [26]. They reported that aPDT mediated by RB, MB, and CUR with white light was effective in killing these microorganisms, although MB and RB were more efficient than CUR. By contrast, our data showed that CUR decreased CFU/mL of E. faecalis by 99.6%. It should be noted that the differences in the results might be due to differences in the microorganisms, concentration of the PSs, irradiation source, and irradiation time. On the other hand, our data showed that CUR-aPDT had a stronger antibiofilm compound potential than aPDT mediated by other PSs. CUR-aPDT reduced the biofilm formation of E. faecalis by about 70%. It has been suggested that it can penetrate into and disrupt E. faecalis biofilms by eliminating the exopolysaccharides matrix of the bacteria [24]. According to a study by
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Neelakantan et al., CUR-aPDT reduces the biofilm formation of E. faecalis on tooth substrate more than 2% chlorhexidine [27]. Despite the marked antimicrobial and antibiofilm properties of CUR, its poor solubility and hydrolytic instability limit its clinical use. Recently Jiang et al. demonstrated that CUR encapsulated into solid lipid nanoparticles could enhance the phototoxic effects of CUR [28]. Although ICG-aPDT could decrease the cell count (98.2%) and biofilm formation (68.4%) of E. faecalis, its activities were less than CUR. One of the important advantages of CUR mediated aPDT for endodontic infection treatment is that it does not change the tooth color or the color of aesthetic materials after treatment [29]. In this regard, some PSs such as TBO and MB can make changes in the color of the tooth surface and resinous restorations due to their highly of pigmented nature [30]. Therefore, CUR can be a good PS for use in aPDT for treatment of endodontic infections, if in vivo evaluations confirm the clinical relevance of these results. The results of the current in vitro study should be interpreted with caution because in vitro assays cannot actually model clinical situations of endodontic infections. One limitation of the current in vitro study was that a single standard strain of E. faecalis was used in the assays in contrast to heterogeneity in antimicrobial resistance, virulence, and genetics of clinical isolates of E. faecalis. Thus, CUR-aPDT that is more effective in vitro may not be effective against the same microorganisms in vivo. Another limitation of this investigation was that culture media instead of microenvironments of root system was used as the niche, which may not mimic the complex dental root environment. It is well known that the contents and properties of the root system during endodontic infections are quite different from those of the culture media, and these differences could potentially have affected the effectiveness of aPDT.
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5. Conclusions In conclusion, our results showed that CUR- and ICG-mediated aPDT had a great antimicrobial and anti-biofilm potential against E. faecalis. TBO mediated aPDT showed considerably greater effects against E. faecalis than MB- aPDT.
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Funding This project was supported by Lorestan University of Medical Sciences, Lorestan, Iran [project no. A_10_1617_2].
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Figure legends: Figure 1. Flowchart of experiment steps.
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Figure 2. Effect of PS alone, irradiation alone, and aPDT against Enterococcus faecalis. Control: bacterial suspension, LED: light emitting diode, aPDT: antimicrobial photodynamic therapy, CFU/mL: colony forming unites per milliliter, ICG: indocyanine green, MB: methylene blue, TBO: toluidine blue O, CUR: curcumin. *Significantly different from control (no treatment), P < 0.05
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Figure 3. Images of plates in each treated group. a. Control, b. Laser 660 nm, c. Laser 635 nm, d. Laser 810 nm, e. Indocyanine green (ICG), f. Light emitting diode (LED), g. Methylene blue (MB), h. Toluidine blue O (TBO), i. Curcumin (CUR), j. Antimicrobial photodynamic therapy with methylene blue (MB-aPDT), k. Antimicrobial photodynamic therapy with toluidine blue O (TBOaPDT), l. Antimicrobial photodynamic therapy with indocyanine green (ICG-aPDT), and m. Antimicrobial photodynamic therapy with curcumin (CUR-aPDT).
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Figure 4. Antibiofilm formation activity of PS alone, irradiation alone, and aPDT against Enterococcus faecalis. LED: light emitting diode, aPDT: antimicrobial photodynamic therapy, ICG: indocyanine green, MB: methylene blue, TBO: toluidine blue O, CUR: curcumin. *Significantly different from control (no treatment), P < 0.05
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Figure 5. Scanning electron microscope images of Enterococcus faecalis biofilm in each treated group. a. Control, b. Laser 660 nm, c. Laser 635 nm, d. Laser 810 nm, e. Indocyanine green (ICG), f. Light emitting diode (LED), g. Methylene blue (MB), h. Toluidine blue O (TBO), i. Curcumin (CUR), j. Antimicrobial photodynamic therapy with methylene blue (MB-aPDT), k. Antimicrobial photodynamic therapy with toluidine blue O (TBO-aPDT), l. Antimicrobial photodynamic therapy with indocyanine green (ICG-aPDT), and m. Antimicrobial photodynamic therapy with curcumin (CUR-aPDT).
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Table 1
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Wavelength (nm) Mode Spot of the probe (mm) Power output (mW) Exposure time (min) Energy density (J/cm2)
Light sources Diode laser LED 635 660 810 450 CW CW CW CW 6.39 6.39 6.39 6.40 220 150 200 1000-1400 5 5 2 5 171.87 117.18 32.5 300-420
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Parameters
Table 2. Effects of different treatment on Enterococcus faecalis viability and biofilm form.
LED
MB
TBO
CUR
MB-aPDT
TBO-aPDT
ICG-aPDT
Standard deviation
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CUR-aPDT
2.8
0.16
3.01 ± 0.10 ×105
5.9
0.21
2.95 ± 0.07 ×105
7.8
0.10
2.90 ± 0.03 ×105
9.3
0.08
2.80 ± 0.10 ×105
12.5
0.06
2.69 ± 0.04 ×105
15.9
2.60 ± 0.08 ×105
18.7
24.3
0.05
65.0
0.00
85.3
0.00
96.8
0.00
98.4
0.00
2.42 ± 0.08 ×105 1.12 ± 0.10 ×105
0.47 ± 0.08 ×105
0.10 ± 0.04 ×105
0.05 ± 0.02 ×105
0.07
0.06
Biofilm assay Mean of % of OD ± SD reduction
P value
3.28 ± 0.06
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-
3.16 ± 0.04
3.4
0.18
3.15 ± 0.09
3.8
0.09
3.10 ± 0.06
5.2
0.09
3.04 ± 0.06
7.1
0.08
2.98 ± 0.05
9.0
0.05
2.88 ± 0.07
12.0
0.06
2.68 ± 0.08
18.0
0.06
2.52 ± 0.07
23.0
0.05
1.73 ± 0.08
47.0
0.00
1.34 ± 0.10
59.0
0.00
1.21 ± 0.06
62.9
0.00
1.03 ± 0.08
68.4
0.00
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3.11 ± 0.06 ×105
3.35 3.22 3.27 3.11 3.17 3.20 3.25 3.15 3.06 3.05 3.08 3.17 2.98 3.10 3.05 3.02 3.00 2.98 2.85 2.97 2.82 2.60 2.76 2.68 2.46 2.60 2.51 1.65 1.82 1.72 1.22 1.43 1.38 1.28 1.16 1.20 1.05 1.11 0.95
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ICG
-
OD 570 nm
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Laser 810 nm
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Laser 635 nm
P value
3.2 ± 0.09 ×105
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Laser 660 nm
3.30 3.12 3.20 3.18 3.11 3.05 2.90 3.05 3.10 2.87 2.98 3.00 2.93 2.91 2.86 2.74 2.93 2.75 2.70 2.65 2.73 2.52 2.68 2.61 2.52 2.40 2.35 1.20 1.01 1.16 0.48 0.55 0.38 0.05 0.12 0.14 0.02 0.06 0.07
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Microbial viability assay Mean of % of CFU/mL ± SD a reduction
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S1572-1000(18)30242-4 https://doi.org/10.1016/j.pdpdt.2018.09.014 PDPDT 1253
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
24-7-2018 17-9-2018 24-9-2018
Please cite this article as: Pourhajibagher M, Kazemian H, Chiniforush N, Hosseini N, Pourakbari B, Azizollahi A, Rezaei F, Bahador A, Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during antimicrobial photodynamic therapy, Photodiagnosis and Photodynamic Therapy (2018), https://doi.org/10.1016/j.pdpdt.2018.09.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Exploring different photosensitizers to optimize elimination of planktonic and biofilm forms of Enterococcus faecalis from infected root canal during
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antimicrobial photodynamic therapy
Maryam Pourhajibagher1, Hossein Kazemian2,3, Nasim Chiniforush4, Nava Hosseini5, Babak Pourakbari6, Atefeh Azizollahi7, Faranak Rezaei8*, Abbas Bahador9
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Dental Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 2 Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran. 3 Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 4 Laser Research Center, Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran. 5 Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran. 6 Pediatric Infectious Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran. 7 School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. 8 Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran. 9 Oral Microbiology Laboratory, Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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*Correspondence Address: Franak Rezaei, Ph.D., Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, PO Box:381351698, Khorramabad, Lorestan. Fax: + 986633120150. Email: [email protected] Highlights Curcumin (CUR) and indocyanine green (ICG) mediated antimicrobial photodynamic therapy (aPDT) have considerable antibacterial effects against Enterococcus faecalis. aPDT by CUR and ICG has more anti-biofilm formation activity against E. faecalis than toluidine blue O (TBO) and methylene blue (MB). The antimicrobial activities of CUR mediated aPDT are higher than TBO and MB.
Abstract
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Background: Despite the high success rate of endodontic treatment, failure may occur in some cases. In this case, Enterococcus faecalis is the most common species in endodontic treatment failure and post-treatment apical periodontitis. Therefore, a new adjunctive strategy is needed for the prevention of endodontic infections due to E. faecalis. The aim of the present study was to compare the antimicrobial and anti-biofilm activities of different common photosensitizers (PSs) for use in antimicrobial photodynamic therapy (aPDT) against E. faecalis. Materials and methods: E. faecalis strain ATCC 29212 was used as the tested strain and methylene blue (MB), toluidine blue O (TBO), indocyanine green (ICG), and curcumin (CUR) were used as PSs. Irradiation was carried out using diode laser and light emitting diode (LED) at wavelengths related to the above PSs. Then, antimicrobial and anti-biofilm activities were measured using the microbial viability assay and crystal violet test, respectively. Results: aPDT with using the above PSs significantly decreased the CFU/mL count of E. faecalis compared to the control group (P < 0.05). The killing percentage of E. faecalis via PS mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0% for CUR, ICG, TBO, and MB, respectively. aPDT using the above PSs significantly decreased the biofilm formation ability of E. faecalis compared to the control group (P < 0.05). The biofilm reduction percentage of the PSs was 68.4%, 62.9%, 59.0%, and 47.6% for CUR, ICG, TBO, and MB, respectively. Conclusion: CUR and ICG mediated aPDT exhibited considerably more antimicrobial activity than other PSs, while TBO and MB demonstrated weaker anti-biofilm effects against E. faecalis compared to other PSs. Keywords: Enterococcus faecalis; antimicrobial photodynamic therapy; methylene blue; toluidine blue O; indocyanine green; curcumin
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1. Introduction Despite the high success rate of endodontic treatment, failure may occur in some cases. One of the important causes of endodontic failure is secondary/persistent endodontic infection in the root canal system, maybe due to evasion of disinfectants at the time of root canal treatment obturation [1]. In this regard, Enterococcus faecalis is the causative agent in 20-70% of endodontic failures due to its ability to form biofilms as well as its high resistance to endodontic medicaments [2-5]. Following the emergence of resistant E. faecalis to antimicrobial agents isolated from root canals, prevention of secondary endodontic infection caused by this bacterium has become very complicated [6]. Efforts in this regard have led to the investigation, development, and setting up of novel antimicrobial strategies. Antimicrobial photodynamic therapy (aPDT) is a novel antimicrobial approach. aPDT acts through generation of reactive oxygen species (ROS) in the presence of molecular oxygen by using a combination of a photosensitizer (PS) and light at a specific wavelength. A PS is a nontoxic dye that binds to the outer surface, cell membrane, and DNA of the microorganism, and becomes activated following irradiation with light. aPDT causes bacterial cell death by microbial cell envelope disruption and DNA damage [7]. Many studies have
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demonstrated the antimicrobial properties of aPDT against local infections [8, 9]. Moreover, it has been shown that aPDT not only can be used for killing bacteria, but it can also be applied to reduce the impact of bacterial virulence factors such as biofilm formation and prevent bacterial colonization in the root canal [10]. Different types of PSs have been used for endodontic treatment in recent years [11]. Toluidine blue O (TBO) and methylene blue (MB) are amphiphilic, positively charged PSs that have a low molecular weight. They penetrate into the outer membrane through binding to microbial surface proteins. These PSs have bactericidal effects against both Gram-positive and Gram-negative bacteria. The absorption of TBO and MB peaks at 635 and 660 nm, respectively [11]. Indocyanine green (ICG) is an anionic PS with antimicrobial properties mainly in photothermal versus photochemical therapy and an absorption peak at 810 nm [11]. Curcumin (CUR), another PS, is a phenolic compound capable of absorbing light at 450 nm [12]. To the best of our knowledge, no study has compared the effect of aPDT based on four common types of PSs against bacterial pathogens. Considering the role of E. faecalis as a leading cause of persistent/secondary endodontic infection, the aim of the present study was to compare the potency of antimicrobial and anti-biofilm effects of TBO-, MB-, ICG-, and CUR-aPDT against E. faecalis to find a better endodontic disinfection procedure.
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2. Material and Methods 2.1. Bacterial strain E. faecalis strain ATCC 29212 was obtained from the Iranian Biological Resource Center (Tehran, Iran) and aerobically grown in the fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37 °C until logarithmic growth phase at a final concentration of 1.0 × 106 colony forming units (CFU)/mL, verified by spectrophotometry at 600 nm.
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2.2. Photosensitizers and light sources Based on the results of previous studies, the maximum concentration of PSs that not causing cytotoxicity and tooth coloration was used [8, 13]. Stock solutions of TBO and MB (SigmaAldrich, Steinheim, Germany) were prepared in sterile 0.9% (wt/vol) NaCl at a final concentration of 0.1 mg/mL. A stock solution of ICG was prepared by dissolving ICG powder (Santa Cruz, USA) in 1.0 mL sterile distilled water at a final concentration of 1.0 mg/mL. A stock solution of CUR (Sigma-Aldrich, Steinheim, Germany), at a final concentration of 5.0 mg/mL, was prepared using a non-toxic concentration of dimethyl sulfoxide (0.5%) [13]. Then, all of the stock solutions were sterilized using a 0.22-nm millipore filter and kept in dark conditions before use. A diode laser at wavelengths of 635 nm (Klas DX62, Konftec, Taiwan), 660 nm (Klas DX62, Konftec, Taiwan), and 810 nm (Klas DX82, Konftec, Taiwan) was used for TBO, MB, and ICG, respectively. A light emitting diode (LED) (DY400-4, Denjoy, China) at a wavelength of 450 nm was applied for activation of CUR (Table 1). According to previous studies, the maximum light exposure time not toxic for eukaryotic cells was used in this study (i.e. 2, 5, 5, and 5 minutes with an energy density of 32.5, 117.18, 171.87, and
300-420 J/cm2 at a wavelength 810, 660, 635, and 450 nm for ICG, MB, TBO, and CUR, respectively; Table 1) [8, 13]. The flowchart of the experiment steps is shown in Figure 1.
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2.3. Study design To evaluate the antimicrobial and anti-biofilm activities of different PSs for use in aPDT against E. faecalis, the experimental groups containing E. faecalis strains (n=39) were subjected to the following: A: PS alone: A1: TBO (n=3) A2: MB (n=3) A3: ICG (n=3) A4: CUR (n=3) B: Radiation alone B1: Diode laser at 635 nm (n=3) B2: Diode laser at 660 nm (n=3) B3: Diode laser 810nm (n=3) B4: LED (n=3) C: aPDT (PSs plus corresponding light) C1: TBO plus diode laser at 635 (n=3) C2: MB plus diode laser at 660 (n=3) C3: ICG plus diode laser 810 (n=3) C4: CUR plus LED (n=3) D: Control group: Bacterial suspension with no treatment (n=3)
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2.4. Evaluation of aPDT against E. faecalis in planktonic form In PS alone groups, 100 µL of the 1.5×106 CFU/mL bacterial suspension was added to each well of a 96–well flat-bottomed microtiter plate (TPP, Trasadingen, Switzerland). Then, 100 µL of each PS was added to the bacterial suspension separately. For bacterial cells to absorb the photosensitizer, the mixture was incubated at room temperature (25 ± 2 oC) in the dark for 5 min. In the radiation alone group, 200 µL of the 1.5×106 CFU/mL bacterial suspension was added to each well of the microtiter plate. The bacterial suspension was then exposed to the light source according to the wavelengths mentioned earlier. aPDT was carried out according to a previous study [8]. Briefly, 100 μL of the 2X BHI broth was added into each well of 96-well flat-bottomed sterile polystyrene microplates. Then, 100 μL of each PS solution was added to each well using a multi-channel pipet. In the next step, 100 μL of the 1.5×106 CFU/mL bacterial suspension was added to each well. The microplates were incubated in a dark room for 5 min. Then, the bacterial suspensions were exposed to radiation corresponding to each PS. In this regard, radiation was done at room temperature (25 ± 2 °C) for 2, 5, 5, and 5 min at 810, 660, 635, and 450 nm for ICG, MB, TBO, and CUR, respectively. A bacterial suspension without any treatment was used as the control group.
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The laser and LED probes were fixed 1 mm above the top surface of the microplate by a microphone stand. To prevent beam reflection from the tabletop during aPDT, a black paper was used under the microplates. Then, after aPDT, for determination of CFU/mL of the bacterial cells, 10 μL aliquots of each well was cultured on BHI agar plates (Merck, Darmstadt, Germany) and incubated at 37 °C for 24 h. The number of CFUs/mL was determined after incubation according to a method proposed by Miles and Misra [14].
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2.5. Evaluation of aPDT against biofilm formation ability of E. faecalis To evaluate the anti-biofilm activities of aPDT mediated by the aforementioned PSs, biofilms of E. faecalis were formed using 12-well flat-bottomed sterile polystyrene microplates according to previous studies with minor modifications [15, 16]. Briefly, 2000 μL of the 1.5 × 108 CFU/mL bacterial suspension was treated as mentioned earlier. Aliquots of the bacterial suspension were grown on polyester coverslips (Thermo Scientific™ Nunc™ Thermanox™, GmbH, Germany) at 37 °C for 24 h. The coverslips were transferred to the new sterile microplates and gently washed using 2000 μL phosphate-buffered saline (PBS, pH: 7.4). In the new sterile microplates, each coverslip was then stained with 0.1% (w/v) crystal violet for 20 min at room temperature, and then washed twice with distilled water. The biofilm was solubilized using 1000 µL of 95% ethanol for 20 min. Then, 100 µL of the content of each well was transferred onto 96-well flat-bottomed sterile polystyrene microplates, and the optical density of each well was quantified at 570 nm using a microplate reader (Thermo Fisher Scientific, US). A bacterial suspension without any treatment was used as the control group.
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2.6. Statistical analysis The results were analyzed with one-way analysis of variance (ANOVA) and Tukeys’ test using the SPSS software version 22.0. All experiments were performed in triplicate. The results are reported as mean ± standard deviation (SD). P values less than 0.05 were considered significant.
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3. Results As shown in Figure 2, aPDT mediated by the aforementioned PSs significantly reduced CFU/mL count of E. faecalis compared to the control group (P < 0.05). CUR, ICG, TBO, and MB mediated aPDT decreased the CFU/mL by 250, 56, 7, and 3 folds compared to the control group, respectively. The killing percentage of E. faecalis by CUR, ICG, TBO, and MB mediated aPDT was 99.6%, 98.2%, 85.1%, and 65.0%, respectively. According to our data, CUR-aPDT reduced the CFU/mL count of the E. faecalis almost equal to ICG-aPDT but more than TBO- and MBaPDT (P < 0.05, Figs. 2 and 3). According our data in Table 2, treatment with each PS (MB, TBO, ICG, and CUR), and radiation alone (diode laser at 635, 660, and 810 nm as well as LED) caused no significant CFU/mL reduction in E. faecalis. In this regard, CUR and ICG mediated aPDT had more antimicrobial activity than TBO- and MB- aPDT. The biofilm reduction percentage of CUR, ICG, TBO and MB mediated aPDT was 68.4%, 62.9%, 59.0%, and 47.6%, respectively (P < 0.05; Fig. 4). Based on the results of SEM images, aPDT mediated by the above PSs disrupted the biofilm structure of E. faecalis more than other groups
(Fig. 5). In addition, as shown in Figure 4, 5 and Table 2, treatment with a PS alone, diode laser alone at different wavelengths, and LED alone caused no significant reduction in the formation of E. faecalis biofilms.
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4. Discussions aPDT is considered a minimally invasive method for treatment of local infections, such as endodontic infection [17, 18]. In the present study, the efficacy of aPDT mediated by different common PSs against planktonic and biofilm growth of E. faecalis was tested. E. faecalis was selected as the test strain because it has been found to colonize in the root canal and cause different disorders [19, 20]. Pourhajibagher et al. showed that ICG decreased the cell count and biofilm formation of Porphyromonas gingivalis to 30.4% and 25.1%, respectively [21]. In the present study, TBO showed high yet non-significant antimicrobial and anti-biofilm activities, more than MB, in PS mediated aPDT against E. faecalis. Our data showed that the killing percentage of E. faecalis by TBO mediated aPDT was 85.1%. The results of this study showed that neither LED nor diode laser nor PSs, when used alone, had antimicrobial and anti-biofilm properties, which was consistent with the results of studies conducted by Pourhajibagher et al. [8], López-Jiménez et al. [22], and Rödig et al. [23]. CUR had more (but non-significant) antimicrobial and anti-biofilm formation activities compared to other PSs. It has been reported that there is an assembly of a protein-filamenting temperaturesensitive mutant Z (FtsZ). The proto-filaments are inhibited by CUR, which leads to an increase in the GTPase activity of FtsZ. Increased GTPase activity due to the FtsZ assembly causes bacterial cell death [24]. Our data showed that CUR-aPDT (5.0 mg/mL, 5 min radiation) had the highest bactericidal and anti-biofilm activities against E. faecalis. The antimicrobial activity of CUR mediated aPDT has been evaluated in different bacterial species. Paschoal et al. showed that simultaneous use of blue light LED plus CUR had a better antimicrobial effect on Streptococcus mutans, which was consistent with our results [25]. Najafi et al. reported that CUR is an effective substance in preventing the growth of Aggregatibacter actinomycetemcomitans, the impact of which is reinforced when used simultaneously with an aPDT method [13]. However, some studies have reported different results compared to our analysis. Lozano et al. evaluated the photodynamic effect of MB, Rose Bengal (RB), and CUR in combination with white light against S. mutans, S. sanguis, and Candida albicans [26]. They reported that aPDT mediated by RB, MB, and CUR with white light was effective in killing these microorganisms, although MB and RB were more efficient than CUR. By contrast, our data showed that CUR decreased CFU/mL of E. faecalis by 99.6%. It should be noted that the differences in the results might be due to differences in the microorganisms, concentration of the PSs, irradiation source, and irradiation time. On the other hand, our data showed that CUR-aPDT had a stronger antibiofilm compound potential than aPDT mediated by other PSs. CUR-aPDT reduced the biofilm formation of E. faecalis by about 70%. It has been suggested that it can penetrate into and disrupt E. faecalis biofilms by eliminating the exopolysaccharides matrix of the bacteria [24]. According to a study by
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Neelakantan et al., CUR-aPDT reduces the biofilm formation of E. faecalis on tooth substrate more than 2% chlorhexidine [27]. Despite the marked antimicrobial and antibiofilm properties of CUR, its poor solubility and hydrolytic instability limit its clinical use. Recently Jiang et al. demonstrated that CUR encapsulated into solid lipid nanoparticles could enhance the phototoxic effects of CUR [28]. Although ICG-aPDT could decrease the cell count (98.2%) and biofilm formation (68.4%) of E. faecalis, its activities were less than CUR. One of the important advantages of CUR mediated aPDT for endodontic infection treatment is that it does not change the tooth color or the color of aesthetic materials after treatment [29]. In this regard, some PSs such as TBO and MB can make changes in the color of the tooth surface and resinous restorations due to their highly of pigmented nature [30]. Therefore, CUR can be a good PS for use in aPDT for treatment of endodontic infections, if in vivo evaluations confirm the clinical relevance of these results. The results of the current in vitro study should be interpreted with caution because in vitro assays cannot actually model clinical situations of endodontic infections. One limitation of the current in vitro study was that a single standard strain of E. faecalis was used in the assays in contrast to heterogeneity in antimicrobial resistance, virulence, and genetics of clinical isolates of E. faecalis. Thus, CUR-aPDT that is more effective in vitro may not be effective against the same microorganisms in vivo. Another limitation of this investigation was that culture media instead of microenvironments of root system was used as the niche, which may not mimic the complex dental root environment. It is well known that the contents and properties of the root system during endodontic infections are quite different from those of the culture media, and these differences could potentially have affected the effectiveness of aPDT.
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5. Conclusions In conclusion, our results showed that CUR- and ICG-mediated aPDT had a great antimicrobial and anti-biofilm potential against E. faecalis. TBO mediated aPDT showed considerably greater effects against E. faecalis than MB- aPDT.
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Funding This project was supported by Lorestan University of Medical Sciences, Lorestan, Iran [project no. A_10_1617_2].
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21. M. Pourhajibagher, N. Chiniforush, R. Ghorbanzadeh, A. Bahador, Photo-activated disinfection based on indocyanine green against cell viability and biofilm formation of Porphyromonas gingivalis, Photodiagnosis. Photodyn. Ther. 17 (2017) 61-64. 22. L. López-Jiménez, E. Fusté, B. Martínez-Garriga, J. Arnabat-Domínguez, T. Vinuesa, M. Viñas, Effects of photodynamic therapy on Enterococcus faecalis biofilms, Lasers. Med. Sci. 30 (2015) 1519-1526. 23. T. Rödig, S. Endres, F. Konietschke, O. Zimmermann, H.G. Sydow, A. Wiegand, Effect of fiber insertion depth on antibacterial efficacy of photodynamic therapy against Enterococcus faecalis in root canals, Clin. Oral. Investig. 21 (2017) 1753-1759.
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24. D. Rai, J.K. Singh, N. Roy, D. Panda, Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity, Biochem. J. 410 (208) 147-155.
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25. M.A. Paschoal, C.C. Tonon, D.M. Spolidório, V.S. Bagnato, J.S. Giusti, L. Santos-Pinto, Photodynamic potential of curcumin and blue LED against Streptococcus mutans in a planktonic culture, Photodiagnosis. Photodyn. Ther. 10 (2013) 313-319.
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26. P. Soria-Lozano, Y. Gilaberte, M.P. Paz-Cristobal, L. Pérez-Artiaga, V. Lampaya-Pérez, J. Aporta, et al., In vitro effect photodynamic therapy with differents photosensitizers on cariogenic microorganisms, BMC. Microbiol. 15 (2015) 187-193.
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27. P. Neelakantan, C. Subbarao, S. Sharma, C.V. Subbarao, F. Garcia-Godoy, J.L. Gutmann, Effectiveness of curcumin against Enterococcus faecalis biofilm, Acta. Odontol. Scand. 71 (2013):1453-1457.
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28. S. Jiang, R. Zhu, X. He, J. Wang, M. Wang, Y. Qian, et al., Enhanced photocytotoxicity of curcumin delivered by solid lipid nanoparticles, Int. J. Nanomedicine. 12 (2016)167-178.
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29. H.A. Ricci Donato, S. Pratavieira, C. Grecco, A. Brugnera-Júnior, V.S. Bagnato, C. Kurachi, Clinical comparison of two photosensitizers for oral cavity decontamination, Photomed. Laser. Surg. 35 (2017) 105-110.
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30. L.N. Dovigo, A.C. Pavarina, J.C. Carmello, A.L. Machado, I.L. Brunetti, V.S. Bagnato, Susceptibility of clinical isolates of Candida to photodynamic effects of curcumin, Lasers. Surg. Med. 43 (2011) 927-934.
Figure legends: Figure 1. Flowchart of experiment steps.
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Figure 2. Effect of PS alone, irradiation alone, and aPDT against Enterococcus faecalis. Control: bacterial suspension, LED: light emitting diode, aPDT: antimicrobial photodynamic therapy, CFU/mL: colony forming unites per milliliter, ICG: indocyanine green, MB: methylene blue, TBO: toluidine blue O, CUR: curcumin. *Significantly different from control (no treatment), P < 0.05
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Figure 3. Images of plates in each treated group. a. Control, b. Laser 660 nm, c. Laser 635 nm, d. Laser 810 nm, e. Indocyanine green (ICG), f. Light emitting diode (LED), g. Methylene blue (MB), h. Toluidine blue O (TBO), i. Curcumin (CUR), j. Antimicrobial photodynamic therapy with methylene blue (MB-aPDT), k. Antimicrobial photodynamic therapy with toluidine blue O (TBOaPDT), l. Antimicrobial photodynamic therapy with indocyanine green (ICG-aPDT), and m. Antimicrobial photodynamic therapy with curcumin (CUR-aPDT).
A
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Figure 4. Antibiofilm formation activity of PS alone, irradiation alone, and aPDT against Enterococcus faecalis. LED: light emitting diode, aPDT: antimicrobial photodynamic therapy, ICG: indocyanine green, MB: methylene blue, TBO: toluidine blue O, CUR: curcumin. *Significantly different from control (no treatment), P < 0.05
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Figure 5. Scanning electron microscope images of Enterococcus faecalis biofilm in each treated group. a. Control, b. Laser 660 nm, c. Laser 635 nm, d. Laser 810 nm, e. Indocyanine green (ICG), f. Light emitting diode (LED), g. Methylene blue (MB), h. Toluidine blue O (TBO), i. Curcumin (CUR), j. Antimicrobial photodynamic therapy with methylene blue (MB-aPDT), k. Antimicrobial photodynamic therapy with toluidine blue O (TBO-aPDT), l. Antimicrobial photodynamic therapy with indocyanine green (ICG-aPDT), and m. Antimicrobial photodynamic therapy with curcumin (CUR-aPDT).
D
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EP
CC
A
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U
N
A
M
D
TE
EP
CC
A
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U
N
A
M
D
TE
EP
CC
A
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U
N
A
M
D
TE
EP
CC
A
SC RI PT
U
N
A
M
D
TE
EP
CC
A
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U
N
A
M
Table 1
A
CC
EP
TE
D
M
A
N
U
Wavelength (nm) Mode Spot of the probe (mm) Power output (mW) Exposure time (min) Energy density (J/cm2)
Light sources Diode laser LED 635 660 810 450 CW CW CW CW 6.39 6.39 6.39 6.40 220 150 200 1000-1400 5 5 2 5 171.87 117.18 32.5 300-420
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Parameters
Table 2. Effects of different treatment on Enterococcus faecalis viability and biofilm form.
LED
MB
TBO
CUR
MB-aPDT
TBO-aPDT
ICG-aPDT
Standard deviation
A
a
CC
CUR-aPDT
2.8
0.16
3.01 ± 0.10 ×105
5.9
0.21
2.95 ± 0.07 ×105
7.8
0.10
2.90 ± 0.03 ×105
9.3
0.08
2.80 ± 0.10 ×105
12.5
0.06
2.69 ± 0.04 ×105
15.9
2.60 ± 0.08 ×105
18.7
24.3
0.05
65.0
0.00
85.3
0.00
96.8
0.00
98.4
0.00
2.42 ± 0.08 ×105 1.12 ± 0.10 ×105
0.47 ± 0.08 ×105
0.10 ± 0.04 ×105
0.05 ± 0.02 ×105
0.07
0.06
Biofilm assay Mean of % of OD ± SD reduction
P value
3.28 ± 0.06
-
-
3.16 ± 0.04
3.4
0.18
3.15 ± 0.09
3.8
0.09
3.10 ± 0.06
5.2
0.09
3.04 ± 0.06
7.1
0.08
2.98 ± 0.05
9.0
0.05
2.88 ± 0.07
12.0
0.06
2.68 ± 0.08
18.0
0.06
2.52 ± 0.07
23.0
0.05
1.73 ± 0.08
47.0
0.00
1.34 ± 0.10
59.0
0.00
1.21 ± 0.06
62.9
0.00
1.03 ± 0.08
68.4
0.00
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3.11 ± 0.06 ×105
3.35 3.22 3.27 3.11 3.17 3.20 3.25 3.15 3.06 3.05 3.08 3.17 2.98 3.10 3.05 3.02 3.00 2.98 2.85 2.97 2.82 2.60 2.76 2.68 2.46 2.60 2.51 1.65 1.82 1.72 1.22 1.43 1.38 1.28 1.16 1.20 1.05 1.11 0.95
U
ICG
-
OD 570 nm
N
Laser 810 nm
-
A
Laser 635 nm
P value
3.2 ± 0.09 ×105
M
Laser 660 nm
3.30 3.12 3.20 3.18 3.11 3.05 2.90 3.05 3.10 2.87 2.98 3.00 2.93 2.91 2.86 2.74 2.93 2.75 2.70 2.65 2.73 2.52 2.68 2.61 2.52 2.40 2.35 1.20 1.01 1.16 0.48 0.55 0.38 0.05 0.12 0.14 0.02 0.06 0.07
EP
Control
Microbial viability assay Mean of % of CFU/mL ± SD a reduction
D
CFU/mL ×
105
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Groups